Many physiological and pathophysiological phenomena, including aging, ischemia/hypoxia, diabetes and some neurodegenerative diseases, promote oxidation of cellular constituents. Methionine and cysteine residues in proteins are readily oxidized; often inducing marked changes in protein function. It is hypothesized that reversible oxidation of cysteine and methionine serves as a physiological modulatory mechanism to regulate protein function. To test this hypothesis, the research program proposed here will examine how oxidation of cysteine and methionine alters gating behavior of human large-conductance calcium-dependent potassium channels (hSIo channels) using electrophysiological assays in combination with molecular mutagenesis. The proposed project will examine how hSIo channel gating is altered by methionine and cysteine oxidation. The biophysical and molecular targets of cysteine and methionine oxidation will be identified. The research program will also study regulation of the hSIo channel by heme, nitric oxide and hypoxia. It is hypothesized that these physiologically relevant variables alter the hSIo channel function in part by cysteine and/or methionine oxidation. Many of the experiments will be conducted in heterologous expression systems so that potential confounding variables, such as the channel subunit composition, are better controlled. These results will be confirmed by using native channels in hippocampal and cortical neurons. The electrophysiological results are quantitatively analyzed to elucidate which specific gating transitions are altered by oxidation of cysteine/methionine and by application of heme/nitric oxide. Previous studies using native calcium-dependent potassium channels often produced conflicting results. The results expected from this research program will clarify many of the important issues raised and provide molecular and biophysical insights into oxidative regulation of ion channels.